Radical copper oxidases are emerging as a broad family of enzymes spanning deep biological divisions. The defining feature of these enzymes, which include the fungal enzymes galactose oxidase and glyoxal oxidase, is the presence of a metalloradical complex comprised of a copper ion and an intrinsic redox cofactor. The protein-derived cofactor, tyrosyl-cysteine (Tyr-Cys) is formed by covalent cross-linking amino acid side chains and undergoes reversible oxidation to a stable protein free radical that plays an essential role in catalysis. This proposal aims to elucidate the mechanism of Tyr-Cys cofactor biogenesis in galactose oxidase, using a combination of Chemical Quench (CQ), Rapid Freeze Quench (RFQ), isotope kinetics and spin trapping experiments. Mass spectrometry will be used to define the sites and structures of covalent protein modifications. Auxotrophic strains of Pichia pastoris will be used to express galactose oxidase incorporating non-natural amino acids as probes of cofactor biogenesis and metalloradical mechanisms. Deuterium labeling of the Tyr-Cys cofactor in the protein will permit the unpaired electron distribution in the Tyr-Cys free radical to be mapped using EPR spectroscopy. Computational experiments on the Tyr-Cys free radical site will provide insight into the properties of this unique catalytic feature. Radical copper oxidases from selected model organisms in widely divergent kingdoms will be expressed and characterized to define the range and roles of this unusual structure in biology. This work will resolve important elements of metalloradical structure and mechanism, and, in a larger context, will contribute to understanding mechanisms of metal-mediated protein modification, a biochemical process that is a necessary step in the maturation of important enzymes including cytochrome c oxidase.